Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein
Copyright © 2001 by Wiley-Liss, Inc.
ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
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Restriction Endonucleases
Derek Robinson, Paul R. Walsh, and Joseph A. Bonventre
Background Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Which Restriction Enzymes Are Commercially
Available? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Why Are Some Enzymes More Expensive Than
Others? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What Can You Do to Reduce the Cost of Working with
Restriction Enzymes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
If You Could Select among Several Restriction Enzymes
for Your Application, What Criteria Should You Consider
to Make the Most Appropriate Choice? . . . . . . . . . . . . . .
What Are the General Properties of Restriction
Endonucleases? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What Insight Is Provided by a Restriction Enzyme’s Quality
Control Data? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How Stable Are Restriction Enzymes? . . . . . . . . . . . . . . . . . .
How Stable Are Diluted Restriction Enzymes? . . . . . . . . . . .
Simple Digests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
How Should You Set up a Simple Restriction Digest? . . . . .
Is It Wise to Modify the Suggested Reaction
Conditions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Complex Restriction Digestions . . . . . . . . . . . . . . . . . . . . . . . . .
How Can a Substrate Affect the Restriction Digest? . . . . .
Should You Alter the Reaction Volume and DNA
Concentration? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Double Digests: Simultaneous or Sequential? . . . . . . . . . . . .
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Genomic Digests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
When Preparing Genomic DNA for Southern Blotting,
How Can You Determine If Complete Digestion
Has Been Obtained? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What Are Your Options If You Must Create Additional
Rare or Unique Restriction Sites? . . . . . . . . . . . . . . . . . . .
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
What Can Cause a Simple Restriction Digest to Fail? . . . .
The Volume of Enzyme in the Vial Appears Very Low. Did
Leakage Occur during Shipment? . . . . . . . . . . . . . . . . . . . .
The Enzyme Shipment Sat on the Shipping Dock for
Two Days. Is It still Active? . . . . . . . . . . . . . . . . . . . . . . . . .
Analyzing Transformation Failure and Other Multiple-Step
Procedures Involving Restriction Enzymes . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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BACKGROUND INFORMATION
Molecular biologists routinely use restriction enzymes as key
reagents for a variety of applications including genomic mapping,
restriction fragment length polymorphism (RFLP) analysis, DNA
sequencing, and a host of recombinant DNA methodologies. Few
would argue that these enzymes are not indispensable tools for
the variety of techniques used in the manipulation of DNA, but
like many common tools that are easy to use, they are not always
applied as efficiently and effectively as possible. This chapter
focuses on the biochemical attributes and requirements of restriction enzymes and delivers strategies to optimize their use in
simple and complex reactions.
Which Restriction Enzymes Are Commercially Available?
While as many as six to eight types of restriction endonucleases
have been described in the literature, Class II restriction endonucleases are the best known, commercially available and the most
useful. These enzymes recognize specific DNA sequences and
cleave each DNA strand to generate termini with 5¢ phosphate
and 3¢ hydroxyl groups. For the vast majority of enzymes characterized to date within this class, the recognition sequence is normally four to eight base pairs in length and palindromic. The point
of cleavage is within the recognition sequence. A variation on this
theme appears in the case of Class IIS restriction endonucleases.
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These recognize nonpalindromic sequences, typically four to
seven base pairs in length, and the point of cleavage may vary
from within the recognition sequence up to 20 base pairs away
(Szybalski et al., 1991).
To date, nearly 250 unique restriction specificities have been
discovered (Roberts and Macelis, 2001). New prototype activities are continually being discovered. The REBASE database
(http://rebase.neb.com) provides monthly updates detailing new
recognition specificities as well as commercial availability.
These enzymes naturally occur in thousands of bacterial strains
and presumably function as the cell’s defense against bacteriophage DNA. Nomenclature for restriction enzymes is based on a
convention using the first letter of the genus and the first two
letters of the species name of the bacteria of origin. For example,
SacI and SacII are derived from Streptomyces achromogenes. Of
the bacterial strains screened for these enzymes to date, well over
two thousand restriction endonucleases have been identified—
each recognizing a sequence specificity defined by one of the
prototype activities. Restriction enzymes isolated from distinct
bacterial strains having the same recognition specificity are known
as isoschizomers (e.g., SacI and SstI). Isoschizomers that cleave
the same DNA sequence at a different position are known as
neoschizomers (e.g., SmaI and XmaI).
Why Are Some Enzymes More Expensive Than Others?
The distribution of list prices for any given restriction enzyme
can vary among commercial suppliers. This is due to many factors
including the cost of production, quality assurance, packaging,
import duties, and freight. For many commonly available enzymes
produced from native overexpressors or recombinant sources,
the cost of production is relatively low and is generally a minor
factor in the final price. Recombinant enzymes (typically overexpressed in a well-characterized E. coli host strain) are often
less expensive than their nonrecombinant counterparts due to
high yields and the resulting efficiencies in production and purification. In contrast, those enzyme preparations resulting in very
low yields are often difficult to purify, and they have significantly
higher production costs. In general, these enzymes tend to be dramatically more expensive (per unit of activity) than those isolated
from the more robust sources. As these enzymes may not be available at the same unit activity levels of the more common enzymes,
they can be less forgiving in nonoptimal reaction conditions,
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and can be more problematic with initial use. The important
point is that the relative price of a given restriction enzyme (or
isoschizomer) may not be the best barometer of its performance
in a specific application or procedure. The enzyme with the highest
price does not necessarily guarantee optimal performance; nor
does the one with the lowest price consistently translate into the
best value.
Most commercial suppliers maintain a set of quality assurance
standards that each product must pass in order to be approved
for release.These standards are typically described in the supplier’s
product catalogs and detailed in the Certificate of Analysis. When
planning to use an enzyme for the first time, it is important to
review the corresponding quality control specifications and any
usage notes regarding recommended conditions and applications.
What Can You Do to Reduce the Cost of Working with
Restriction Enzymes?
Most common restriction enzymes are relatively inexpensive
and often maintain full activity past the designated expiration
date. Restriction enzymes of high purity are often stable for many
years when stored at -20°C. In order to maximize the shelf life of
less stable enzymes, many laboratories utilize insulated storage
containers to mitigate the effects of freezer temperature fluctuations. Periodic summary titration of outdated enzymes for activity is another way to reduce costs for these reagents. For most
applications, 1 ml is used to digest 250 ng to 1 mg of DNA. Enzymes
supplied in higher concentrations may be diluted prior to the reaction in the appropriate storage buffer. A final dilution range of
2000 to 5000 Umits/ml is recommended. However, reducing the
amount of enzyme added to the reaction may increase the risk of
incomplete digestion with insignificant savings in cost. Dilution is
a more practical option when using very expensive enzymes, when
sample DNA concentration is below 250 ng per reaction, or when
partial digestion is required. When planning for partial digestion,
serial dilution (discussed below) is recommended. Most diluted
enzymes should be stable for long periods of time when stored at
-20°C. As a rule it is wise to estimate the amount of diluted
enzyme required over the next week and prepare the dilution in
the appropriate storage buffer, accordingly. For immediate use,
most restriction enzymes can be diluted in the reaction buffer,
kept on ice, and used for the day. Extending the reaction time to
greater than one hour can often be used to save enzyme or ensure
complete digestion.
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If You Could Select among Several Restriction Enzymes for
Your Application, What Criteria Should You Consider to
Make the Most Appropriate Choice?
Each restriction endonuclease is a unique enzyme with individual characteristics, which are usually listed in suppliers’ catalogs
and package inserts. When using an unfamiliar enzyme, these data
should be carefully reviewed. In addition some enzymes provide
additional activities that may impact the immediate or downstream application.
Ease of Use
For many applications it is desirable and convenient to use 1 ml
per reaction. Most suppliers offer standard enzyme concentrations
ranging from 2000 to 20,000 units/ml (2–20 units/ml). In addition
many suppliers also offer these enzymes in high concentration
(often up to 100,000 units/ml), either as a standard product, or
through special order. Enzymes sold at 10 to 20 units/ml are
common and usually lend themselves for use in a wider variety of
applications. When planning to use enzymes available only in
lower concentrations (near 2000 units/ml), be sure to take the final
glycerol concentration and reaction volume into account. By
following the recommended conditions and maintaining the
final glycerol concentration below 5%, you can easily avoid star
activity.
Star Activity
When subjected to reaction conditions at the extremes of their
operating range, restriction endonucleases are capable of cleaving
sequences that are similar, but not identical, to their canonical
recognition sequences. This altered specificity has been termed
“star activity.” Star sites are related to the recognition site, usually
differing by one or more bases. The propensity for exhibiting star
activity varies considerably among restriction endonucleases. For
a given enzyme, star activity will be exhibited at the same relative
level in each lot produced, whether isolated from a recombinant
or a nonrecombinant source.
Star activity was first reported for EcoRI incubated in a low
ionic strength high pH buffer (Polisky et al., 1975). Under these
conditions, while this enzyme would cleave at its canonical site
(G/AATTC), it also recognized and cleaved at N/AATTC. This
reduced specificity should be a consideration when planning to use
a restriction endonuclease in a nonoptimal buffer. It was also
found that substituting Mn2+ for Mg2+ can result in star activity
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(Hsu and Berg, 1978). Prolonged incubation time and high enzyme
concentration as well as elevated levels of glycerol and other
organic solvents tend to generate star activity (Malyguine,
Vannier, and Yot, 1980). Maintaining the glycerol concentration to
5% or less is recommended. Since the enzyme is supplied in 50%
glycerol, the enzyme added to a reaction should be no more than
10% of the final reaction volume.
When extra DNA fragments are observed, especially when
working with an enzyme for the first time, star activity must be
differentiated from partial digestion or contaminating specific
endonucleases. First, check to make sure that the reaction conditions are well within the optimal range for the enzyme. Then,
repeat the digest in parallel reactions, one with twice the activity
and one with half the activity of the initial digest. Partial digestion
is indicated as the cause when the number of bands is reduced
to that expected after repeating the digestion with additional
enzyme (or extending incubation time). If extra bands are still
evident, contact the supplier’s technical support resource for
advice. Generally speaking, star activity and contaminating activities are more difficult to differentiate. Mapping and sequencing
the respective cleavage sites is the best method to distinguish star
activity from a partial digest or contaminant activity.
Site Preference
The rate of cleavage at each site within a given DNA substrate
can vary (Thomas and Davis, 1975). Fragments containing a subset
of sites that are cleaved more slowly than others can result in
partial digests containing lighter bands visualized on an ethidium
stained agarose gel. Certain enzymes such as EcoRII require an
activator site to allow cleavage (Kruger et al., 1988). Substrates
lacking the additional site will be cleaved very slowly. For certain
enzymes (NaeI), adding oligonucleotides containing the site or
adding another substrate containing multiple sites can improve
cutting. In the case of PaeR7I, it has been shown that the surrounding sequence can have a profound effect on the cleavage
rate (Gingeras and Brooks, 1983). In most cases this rate difference is taken in to account because the unit is defined at a point
of complete digestion on a standard substrate DNA (e.g., lambda
DNA) that contains multiple sites. Problems can arise when
certain sites are far more resistant than others, or when highly
resistant sites are encountered on substrates other than the standard substrate DNA. If a highly resistant site is present in a
common cloning vector, then a warning should be noted on the
data card or in the catalog.
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Methylation
Methylation sensitivity can interfere with digestion and cloning
steps. Many of the E. coli cloning strains express the genes for
EcoKI methylase, dam methylase, or dcm methylase. The dam
methylase recognizes GATC and methylates at the N6 position
of adenine. MboI recognizes GATC (the same four base-pair
sequence as dam methylase) and will only cleave DNA purified
from E. coli strains lacking the dam methylase. DpnI is one of only
a few enzymes known to cleave methylated DNA preferentially,
and it will only cleave DNA from dam+ strains (Lacks and Greenberg, 1977). Another E. coli methylase, termed dcm, was found to
block AatI and StuI (Song, Rueter, and Geiger, 1988). The dcm
methylase recognizes CC(A/T)GG and methylates the second C
at the C5 position.
The restriction enzyme recognition site doesn’t have to span the
entire methylation site to be blocked. Overlapping methylation
sites can cause a problem. An example is the XbaI recognition site
5¢ TCTAGA 3¢. Although it lacks the GATC dam methylase
target, if the preceding 5¢ two bases are GA giving GATCTAGA
or the following 3¢ bases are TC giving TCTAGATC, then the dam
methylase blocks XbaI from cutting. E. coli strains with deleted
dam and dcm, like GM2163, are commercially available and
should be used if the restriction site of interest is blocked by
methylation. The first time a methylated plasmid is transformed
into GM2163 the number of colonies will be low due to the important role played by dam during replication.
Methylation problems can also arise when working with mammalian or plant DNA. DNA from mammalian sources contain
C5 methylation at CG sequences. Plant DNA often contains C5
methylation at CG and CNG sequences. Bacterial species contain
a wide range of methylation contributed by their restriction modification systems (Nelson, Raschke, and McClelland, 1993). Information regarding known sensitivities to methylation can be found
on data cards in catalog tables, by searching REBASE, and in the
preceding review by Nelson.
Cloning problems can arise when working with DNA methylated at the C5 position. Most E. coli strains have an mcr restriction system that cleaves methylated DNA (Raleigh et al., 1988).
A strain deficient in this system must be used when cloning DNA
from mammalian and plant sources.
Substrate Effects
More on this discussion appears in the question below, How
Can a Substrate Affect the Restriction Digest?
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WHAT ARE THE GENERAL PROPERTIES OF
RESTRICTION ENDONUCLEASES?
In general, commercial preparations of restriction endonucleases are purified and stored under conditions that ensure optimal
reactivity and stability over time; namely -20°C. They are commonly supplied in a solution containing 50% glycerol, Tris buffer,
EDTA, salt, and reducing agent. This solution will conveniently
remain in liquid form at -20°C but will freeze at temperatures
below -30°C. Those enzymes shipped on dry ice, or stored at
-70°C, will have a white crystalline appearance; they revert to a
clear solution as the temperature approaches -20°C. As a rule
repeated freeze-thaw cycles are not recommended for enzyme
solutions because of the possible adverse effects of shearing (more
on the question, How Stable are Restriction Enzymes? appears
below).
As a group (and by definition), Class II restriction endonucleases require magnesium (Mg2+) as a cofactor in order to cleave
DNA at their respective recognition sites. Most restriction
enzymes are incubated at 37°C, but many require higher or lower
(i.e., SmaI requires incubation at 25°C) temperatures. Percent
activity tables of thermophilic enzymes incubated at 37°C can be
found in some suppliers’ catalogs. For most reactions, the pH
optima is between 7 and 8 and the NaCl concentration between
50 and 100 mM. Concentrated reaction buffers for each enzyme
are provided by suppliers. Typically each enzyme is profiled for
optimal activity as a function of reaction temperature, pH (buffering systems), and salt concentration. Some enzymes are also
evaluated in reactions containing additional components (BSA,
detergents). Generally, these characteristics are documented in
the published literature and referenced by suppliers.
Interestingly, a number of commonly used enzymes can display
a broad range of stability and performance characteristics under
fairly common reaction conditions. They may vary considerably in
activity and may exhibit sensitivity to particular components. In
an effort to minimize these undesirable effects, suppliers often
adjust enzyme buffer components and concentrations to ensure
optimal performance for the most common applications.
There is a wealth of information about the properties of these
enzymes in most suppliers’ catalogs, as well as on their Web sites.
The documentation supplied with the restriction endonuclease
should contain detailed information about the enzyme’s properties and functional purity. It is important to read the Certificate of Analysis when using a restriction enzyme for the first
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time, as it may provide important information concerning particular substrate DNAs or alternative reaction conditions for a specific application.
What Insight Is Provided by a Restriction Enzyme’s
Quality Control Data?
Restriction enzymes are isolated from bacterial strains that
contain a variety of other enzyme activities required for normal
cell function. These additional activities include other nucleases,
phosphatases, and polymerases as well as other DNA binding proteins that may inhibit restriction enzyme activity. In preparations
where trace amounts of these activities remain, the end-structure
of the resulting DNA fragments may be degraded, thus inhibiting
subsequent ligation. Likewise plasmid substrates may be nicked,
thus reducing transformation efficiencies.
Ideally the restriction enzyme preparation should be purified to
homogeneity and free of any detectable activities that might interfere with digestion or inhibit subsequent reactions planned for the
resulting DNA fragments. In order to provide researchers with a
practical means to conveniently evaluate the suitability of a given
restriction enzyme preparation, suppliers include a Certificate
of Analysis with each product, detailing the preparation’s performance in a defined set of Quality Control Assays. In order to
establish a standard reference for the amount of enzyme and substrate used in these assays, each supplier must first define the unit
substrate and reaction conditions for each product.
Unit Definition
A unit of restriction endonuclease is defined as the amount of
enzyme required to completely cleave 1 mg of substrate DNA suspended in 50 ml of the recommended reaction buffer in one hour
at the recommended assay buffer and temperature. The DNA
most often used is bacteriophage Lambda or another wellcharacterized substrate. Note that the unit definition is not based
on classic enzyme kinetics. The enzyme molar concentration is in
excess. A complete digest is determined by the visualized pattern
of cleaved DNA fragments resolved by electrophoresis on an
ethidium bromide-stained gel. Some restriction enzymes will
behave differently when used outside the parameters of the unit
definition. The number of sites (site density) or the particular type
of DNA substrate may have an effect on “unit activity,” but it is
not always proportional (Fuchs and Blakesley, 1983).
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Quality Control Assays—Maximum Units per Reaction
When using procedures requiring larger quantities of enzyme
and/or extended reaction times, an appreciation of the quality
control data can help determine a safe amount of enzyme for your
application.
Overnight Assay
Increasing amounts of restriction endonuclease are incubated
overnight (typically for 16 hours) in their recommended buffer
with 1 mg of substrate DNA in a volume of 50 ml. The characteristic limit digest banding pattern produced by the enzyme in one
hour is compared to the pattern produced from an excess of
enzyme incubated overnight. A sharp, unaltered pattern under
these conditions is an indication that the enzyme preparation
is free of detectable levels of nonspecific endonucleases. The
maximum number of units yielding an unaltered pattern is
reported. Enzymes listing 100 units or more, a 1600-fold over
digestion (100 units ¥ 16 h), will not degrade DNA up to megabase
size in mapping experiments and can be assumed to be virtually
free of nonspecific endonuclease (Davis, T. and Robinson, D.,
unpublished observations).
Nicking Assay
Another sensitive test for contaminating endonucleases is
a four hour incubation with a supercoiled plasmid that lacks
a site for the enzyme being tested. The supercoil is very sensitive to nonspecific nicking by a single-stranded endonuclease,
cleavage by a double-stranded endonuclease, or topoisomerase
activity. If a single-stranded nick occurs, the supercoiled molecule, RFI, unwinds and assumes the circular form, RFII. If a
double-stranded cleavage occurs, the circle will become linear.
High levels of single-stranded nicking leads to linear DNA.
All three forms of DNA have distinct electrophoretic mobilities
on agarose gels. Enzymes converting 5% or less of the plasmid
to relaxed form using 100 units of enzyme for four hours can
be considered virtually free of nicking activity. High-salt buffers,
especially at elevated temperature, can cause some conversion
to relaxed form. A control reaction, including buffer and DNA
but lacking enzyme, is incubated and run on the agarose gel for
comparison.
Exonuclease Assay
Suppliers use a variety of assays to check for exonuclease activity. A general assay mixture contains a restriction endonuclease
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with 1 mg of a mixture of single- and double-stranded, 3H-labeled
E. coli DNA (200,000 cpm/mg) in a 50 ml reaction volume with the
supplied buffer. Incubations (along with a background control
containing no enzyme) are at the recommended temperature for
four hours. Exonuclease contamination is indicated by the percent
of the total labeled DNA in the reaction that has been rendered
TCA-soluble. The limit of detectability of this assay is approximately 0.05%. Enzymes showing background levels of degradation with 100 units incubated for four hours can be considered
virtually free of exonuclease.
Ligation/Recut Assay
Ligation and recutting is a direct determination of the integrity
of the DNA fragment termini upon treatment with the restriction
enzyme preparation. Ligation and recut of greater than 90% with
a 10- to 20-fold excess of enzyme creating ends with overhangs or
80% for blunt ends indicate an enzyme virtually free of exonuclease or phosphatase specific for the overhang being tested.
Alternative assays (i.e., end-labeling) are used to evaluate Type
IIS restriction enzymes (e.g., FokI, MboII). Since these enzymes
cleave outside of their recognition sequence, the standard ligation
assay would not determine a loss of terminal nucleotides due to
exonuclease. The resulting ends could still ligate, and since their
recognition sites remain intact, the enzyme would still be able to
recut.
Blue-White Screening Assay
The b-galactosidase blue-white selection system is also applied
to determine the integrity of the DNA ends produced after digestion with an excess of enzyme to test ligation efficiency. An intact
gene gives rise to a blue colony; while an interrupted gene, which
contains a deletion due to degraded DNA termini, gives rise to a
white colony. Restriction enzymes tested using this assay should
produce fewer than 3% white colonies.
The values given for the number of units added giving
“virtually contaminant-free” preparations are somewhat arbitrary. They are useful, however, for determining maximum
levels of enzyme to use in a reaction for most common applications. Enzymes with quality control results significantly below
these values can still be used with confidence under simple
assay conditions. As discussed later for complex restriction
digestions, caution should be considered when extending reaction
times and adding more than 1 to 2 ml of enzyme to 1 mg DNA
in 50 ml.
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How Stable Are Restriction Enzymes?
As a class, most restriction enzymes are stable proteins. Even
during purification periods lasting two weeks, many enzymes lose
no appreciable activity at 4°C. At the final stage of purification,
the enzyme preparation is typically dialyzed into a 50% glycerol
storage buffer and subsequently stored at -20°C. At this temperature the glycerol solution does not freeze. Most enzymes are
stable for well over a 12-month period when properly stored. In
one stability test of 170 restriction enzymes, activity was assessed
after storage for 16 hours at room temperature. Of the enzymes
tested, 122 (or 72%) exhibited no loss in activity (McMahon, M.,
and Krotee, S., unpublished observation). This point is important
to note in case of freezer malfunction.
Even under optimal storage conditions, however, some enzymes
may begin to lose noticeable activity within a six-month period.
The supplier’s expiration date, Certificate of Analysis, or catalog
will provide more specific information regarding these enzymes.
It is best to use these enzymes within a reasonable amount of time
after they have been received. Some users employ a freezer box
designed to maintain a constant temperature (for short periods at
the bench) to store enzymes within the freezer. Alternatively, most
enzymes can be stored at -70°C for extended periods. Repeated
freeze–thaw cycles from -70°C to 0°C is not recommended. Each
time the enzyme preparation solution is frozen, the buffer comes
out of solution prior to freezing. As a result some enzymes may
lose significant activity each time a freeze–thaw cycle is repeated.
Often the extent of an enzyme’s stability during storage at -20°C
is buffer-related. Identical enzyme preparations obtained from
two suppliers, when maintained in their respective storage buffers,
may have significantly different shelf lives.
How Stable Are Diluted Restriction Enzymes?
For a discussion, refer above to the question What Can You Do
to Reduce the Cost of Working with Restriction Enzymes.
SIMPLE DIGESTS
How Should You Set up a Simple Restriction Digest?
Reaction Conditions
Most restriction digests are designed either to linearize a
cloning vector or to generate DNA fragments by cutting a given
target DNA to completion at each of the corresponding restriction sites. To ensure success in any subsequent manipulations (i.e.,
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ligation), the enzyme treatment must leave each of the resulting
DNA termini elements intact.
To 1 mg of purified DNA in 50 ml of 1¥ reaction buffer, 1 ml of
enzyme is added and the reaction is incubated for one hour at
the recommended reaction temperature. In most instances the
amount of DNA can be safely varied from about 250 ng to several
micrograms and the volume can be varied between 20 ml and
100 ml. Suitable reaction times may be as little as 15 minutes or
as long as 16 hours. Common DNA purification protocols, as
well as commercially available kits, yield DNA that is suitable for
most digestions. Most commonly used restriction enzymes are of
high purity, inexpensive, and provided at concentrations of 5 to
20 units/ml. Using 1 to 2 ml will overcome any expected variability
in DNA source, quantity, and purity. The length of incubation time
may be decreased to save time or increased to ensure complete
digestion of the last few tenths of a percent of substrate, as the
reaction asymptotically approaches completion.
Control Reactions
Aside from the mere discipline of maintaining “good laboratory
practice,” the ultimate savings realized in time and effort by
running a simple control reaction is often underestimated. Control
reactions can often reveal the cause of a failed digest or point to
the step within a series of reactions responsible for generating
an unexpected result. For every experimental restriction enzyme
reaction set performed, a control reaction (containing sample
DNA, reaction buffer, and no restriction enzyme) should also be
included and analyzed on the agarose gel. Degradation of DNA
in the control reaction may indicate nuclease contamination in the
DNA preparation or in the buffer. The control reaction products
run alongside the sample reaction products on the agarose gel
enables for a more accurate assessment of whether the reaction
went to completion. Running the appropriate size markers is also
recommended.
Is It Wise to Modify the Suggested Reaction Conditions?
Suppliers devote considerable effort in formulating specific
enzyme preparations and the corresponding reaction buffers in
order to ensure sufficient enzyme activity for most common applications. In addition suppliers often provide data (Activity Table)
indicating the relative activity of each enzyme when incubated
under standard reaction conditions for a variety of reaction
buffers provided. This is a useful guide when planning multiple
Restriction Endonucleases
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restriction enzyme digests. For enzymes with low activity in these
standard buffers, specialized buffers are typically supplied.
Restriction enzymes also have a broad range of activity in
nonchloride salt buffers. Some suppliers also offer a potassiumacetate or potassium-glutamate single-buffer system that is formulated to be compatible with a significant subset of their
enzymes. (McClelland et al., 1988; O Farrell, Kutter, and
Nakanishe, 1980). The reaction buffers themselves are typically
supplied as concentrated solutions, ranging from 2¥ to 10¥, and
should be properly mixed upon thawing prior to final dilution.
It is important to note that the reaction buffer supplied with a
given enzyme is the same buffer in which all quality assurance
assays are performed, and documented in the Certificate of Analysis provided. Consequently certain modifications to the recommended reaction conditions (i.e., adding components or changing
reaction volume, temperature, or time of incubation) may produce
unexpected results. Restriction enzymes can vary considerably in
sensitivity to particular changes in their reaction parameters.
While salt concentration may have a significant effect on activity,
salt type (i.e., NaCl vs. KCl) is usually not critical. One exception
would be in the case of SmaI, which has a strong preference for
KCl. For most sensitive enzymes the Certificate of Analysis will
detail any reaction modifications not recommended as well as any
suggestions for alternative reaction conditions. In order to determine whether a given enzyme may be sensitive to an intended
variation in reaction conditions, the Activity Table is also a useful
reference. As a rule the most robust enzymes exhibit high relative
activity across the range of buffers listed (PvuII). Conversely,
those enzymes showing a narrow range for high activity may
require additional consideration prior to any change in reaction
conditions (SalI) and the technical resources provided by the supplier should be consulted.
All restriction enzymes, as do most other nucleases, require
Mg2+ as a cofactor for the DNA cleavage reaction; most buffers
for restriction enzymes contain 10 mM Mg2+. To protect DNA
preparations in storage buffer from any trace nucleases, EDTA (a
Mg2+ chelator) is used, often stocked as a disodium salt solution.
This is commonly used in various stop-dye solutions as well as
electrophoresis buffer. DNA preparations with excessive concentrations of EDTA may inhibit restriction endonuclease cleavage, especially if the DNA solution represents a high proportion
of the final reaction volume. Addition of Mg2+ will alleviate the
inhibition.
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Robinson et al.
A reducing agent, like dithiothreitol or b-mercaptoethanol, is a
frequent buffer component even though it is not required for
enzyme activity. However, as reaction buffers are typically diluted
to their final reaction volume with distilled water, oxidation (i.e.,
from dissolved oxygen) could significantly reduce enzyme activity
in the absence of sufficient reducing agent. BSA is frequently
added as a stabilizing component to restriction enzyme preparations (Scopes, 1982). BSA increases the overall protein concentration and, by coating the hydrophobic surfaces of plastic vials,
prevents possible denaturation. The activity level of many restriction enzymes in a reaction may be significantly enhanced if the
final BSA concentration is around 100 mg/ml. Sometimes non-ionic
detergents, like Triton ¥-100 or Tween 20, are added as stabilizers
for particular enzymes (EcoRI, NotI). A few restriction endonucleases, like BsgI, have their activity significantly increased by the
addition of S-adenosylmethionine (REBASE).
As most restriction enzymes are isolated from mesophilic bacteria, the vast majority exhibit excellent activity at 37°C in a nearneutral pH buffer. An increasing number of enzymes are being
isolated from thermophilic bacteria, which display optimal activity
within the range of 50°C to 75°C. As it happens, a good number of
these enzymes also retain adequate activity at 37°C, and while this
temperature may not be optimal for a particular enzyme, a supplier
may list it as such for convenience in double-digest applications.
COMPLEX RESTRICTION DIGESTIONS
Complex reactions include double digests, reactions using
nonoptimal buffers, reactions with DNA containing sites close to
the ends, reactions with PCR products, and reactions involving
multiple steps. In addition these include reactions with DNA concentrations that are significantly higher or lower than the recommended 1 mg/50 ml as well as simple reactions that simply didn’t
work the first time.
How Can a Substrate Affect the Restriction Digest?
PCR Products
Restriction endonucleases can often be used directly on PCR
products in the PCR reaction mix. Suppliers often provide data
indicating relative enzyme activity under these reaction conditions. Restriction endonuclease activity is influenced by the buffer
used for PCR as well as the enzyme’s ability to cleave in the pres-
Restriction Endonucleases
239
ence of primers. The excess primers present in PCR reactions have
been shown to inhibit SmaI and NdeI (Abrol and Chaudhary,
1993), but many restriction endonucleases can cleave in the presence of a 100-fold molar excess of primers. If your PCR products
were not digested satisfactorily, eliminate the primers by gel purification, desalting column chromatography, membrane filtration or
glass (Bhagwat, 1992).
Ends of Linear Fragments
Restriction endonucleases differ in their ability to cleave at
recognition sites close to the end of a DNA fragment. Cleavage
close to the end of a fragment is important when two restriction
sites are close together in the cloning region of a plasmid and when
cleaving near the ends of PCR products. Many restriction enzymes
can cleave near a DNA end having one base pair in addition to a 1
to 4 single-base overhang produced by an initial cleavage; others
require at least 3 base pairs in addition to an overhang (Moreira
and Noren, 1995).When designing PCR primers containing restriction sites, adding eight random bases 5¢ of the restriction site is recommended for complete digestion of the restriction sites.
Plasmids
Supercoiled plasmids often require more restriction endonuclease to achieve complete digestion than linear DNA. Manufacturers’ catalogs often contain tables listing the number of units
of restriction enzyme required to completely cleave 1 mg of
commonly used supercoiled plasmids.
Inhibitors
Contaminants in the DNA preparation can inhibit restriction
endonuclease activity. Residual SDS from alkaline lysis procedures can inhibit restriction endonucleases. High concentrations
of NaCl, CsCl, other salts, or EDTA can inhibit restriction enzymes. Salt is concentrated when the DNA is alcohol precipitated.
Washes containing 70% alcohol following the initial precipitation
will solubilize some salt, but dialysis is preferred.
Protein contaminants in the DNA preparation can influence
the restriction digests. Double strand specific exonucleases can
co-purify with plasmid DNA when using column purification
procedures (Robinson, D., and Kelley, K., unpublished observation). Phenol chloroform extraction followed by ethanol precipitation is an efficient method of removing proteins from DNA
samples. The phenol and chloroform as well as the alcohol must
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Robinson et al.
be thoroughly removed to ensure restriction enzyme activity.
Residual phenol and chloroform are removed by the alcohol precipitation and 70% alcohol wash steps. Alcohol is removed by desiccation. Dialysis can be used to remove residual alcohol that may
be present from a DNA sample that was resuspended before the
alcohol was completely removed. Alcohol can be introduced as a
wash before elution when using diatomaceous earth as a resin for
DNA purification. The resin must be thoroughly dried before
DNA elution to remove the alcohol.
Core histones present on eukaryotic chromosomes can be
difficult if not impossible to remove. Proteinase K followed by
phenol chloroform extraction is often used in these preparations.
Proteinase K is also used when preparing intact chromosomal
DNA embedded in agarose for megabase mapping by pulse field
gel electrophoresis (PFGE). Proteinase K must be inactivated
using phenol chloroform or PMSF. Since the inhibition of proteinase K by a proteinase inhibitor such as PMSF is reversible,
agarose blocks containing proteinase K should be extensively
washed by changing the buffer multiple times. Most restriction
enzymes are active in solutions containing PMSF.
Should You Alter the Reaction Volume and DNA
Concentration?
Reaction Volume
A standard reaction volume to cleave 1 to 2 mg of DNA is 50 ml.
Caution must be used when decreasing the reaction volume. Star
activity tends to increase with decreasing reaction volume. The
increase is most likely due to the higher glycerol concentration
in the smaller volumes. Using 2 ml of BamHI containing 50%
glycerol in a 10 ml reaction gives a final glycerol concentration of
10%. Increasing the reaction volume is not common unless more
than 1 mg of DNA is being digested. Increasing the volume should
be less problematic than decreasing the volume.
DNA Concentration
Varying the DNA concentration significantly from the standard
(1 mg in 50 ml) can cause problems. Decreasing the amount of
DNA or increasing the amount of overdigestion can increase
star activity. An additional fourfold overdigestion occurs when
250 ngs are digested compared to 1 mg when using the same
number of units of restriction enzyme. Low DNA concentrations
near the Km of a restriction enzyme could inhibit cleavage. The
Km for lambda DNA is 1000-fold less than 1 mg/50 ml (Fuchs &
Restriction Endonucleases
241
Blakesley, 1983). Increasing the amount of DNA in 50 ml in most
cases will not have a negative impact on the reaction. HindIII has
been reported to work more efficiently on higher concentration
DNA (Fuchs & Blakesley, 1983). Increasing the number of units
or length of reaction will make up for the excess DNA. Care must
be taken with the addition of extra enzyme, to keep the glycerol
concentration to less than 5%. When digesting large quantities of
DNA, using a concentrated enzyme is desirable. Inhibition may
become a problem if the DNA has contaminants that influence
enzyme activity. Salt and other contaminants in the DNA solution
are more likely to be problematic if the DNA solution represents
a large percentage of the final reaction mix.
Reaction Time
Extended digestion times can be used to increase the performance of a restriction enzyme, but the stability of the restriction
enzyme in reaction should be checked by consulting the manufacturer’s “survival in reaction” tables. BSA added to 100 mg/ml
can increase survival. One should also consider that any trace
contaminants in the preparation may continue to be active during
an extended reaction. Often lower reaction temperatures can be
used with unstable enzymes to increase performance when used
for extended periods. One Unit of PmeI will digest 1 mg of DNA
in two hours at 37°C but can digest 2 mg lambda in two hours at
25°C (Robinson, D., unpublished observation). When using PmeI
for digesting agarose–embedded DNA, an incubation at 4°C
overnight followed by one to two hours at 37°C is suggested.
Double Digests: Simultaneous or Sequential?
Simultaneous
The most convenient way to produce two different ends is
to cut both at the same time in one reaction mix. Often the conditions for one enzyme or the other is not ideal. Manufacturers’
buffer charts give the percent activity in buffers other than
the one in which the enzyme is titered. If there is a buffer that
indicates at least 50% activity for each enzyme, a coordinated
double digest can be performed. Inexpensive, highly pure enzymes
with no notes warning against star activity can be used in excess
with confidence. A 10- to 20-fold excess of enzyme is recommended to increase the chances of success. Two microliters of
a 10 unit/ml stock will give a 10-fold overdigest when used for
one hour on 1 mg in a buffer giving 50% activity. If the enzyme is
stable in reaction, then incubating for longer periods will increase
242
Robinson et al.
the amount of overdigestion. Consult the manufacturer’s stability
information.
If the reaction produces extra fragments, possibly caused by star
activity, reduce the reaction time or the amount of enzyme. If the
reaction is incomplete, individually test each enzyme to determine
it’s ability to linearize the plasmid. A lack of cutting may indicate
an inactive enzyme, absence of the expected site, or inhibitors in
the template preparation. Test the enzyme on a second target as
a control. If both enzymes are active, and the restriction sites are
within several bases of each other, there may be a problem cutting
close to the end of the fragment.
Sequential
Enzyme sets that are not compatible for double digests require
sequential digestion. Always perform the first digest with the
enzyme requiring the lower salt buffer. Either salt (or the corresponding 10¥ reaction buffer) may then be added to the reaction
and the second enzyme can be used directly. To prevent the first
enzyme from exhibiting star activity in the second buffer, it is wise
to heat inactivate prior to addition of the second enzyme. Addition of BSA, reducing agents, or detergents has no adverse effects
on restriction enzymes and may be safely added as required to the
reaction.
If the pH requirements between the two enzymes differ by
more than 0.5 pH units or the difference in salt requirement is
critical (NaCl vs. KCl), alcohol precipitation between enzyme
treatments is commonly performed. Alternatively, drop dialysis
(see procedure D at the end of this chapter) is an option. A strategy that can often save a dialysis step would be to perform the
first reaction in a 20 ml volume and then add 80 ml containing 10 ml
of the higher salt buffer and enzyme to the initial reaction. The
second reaction approximates the standard conditions for that
enzyme.
Expensive enzymes should be optimized and used first in
sequential reactions. When planning to use enzymes from different suppliers, first consider their optimal activity by looking at the
NaCl or KCl requirements. Compare the buffer charts of both
suppliers to determine if the enzyme is used in a standard or optimized buffer. Enzymes that are sold with optimized buffers should
be used in those buffers when possible. If the same enzyme is sold
by both suppliers, compare the two reaction buffers. Remember,
the enzyme is titered in the buffer that is supplied. One supplier
may choose to improve titer using a detergent and BSA, while the
Restriction Endonucleases
243
other may be using a different salt, pH, or enzyme concentration.
In some cases a supplier may be categorizing an enzyme into a
core buffer system by increasing the molar concentration of the
enzyme. If used in an optimized buffer, this enzyme would titer at
higher activity. If an enzyme from another supplier is used in this
suboptimal core buffer, poor activity may result.
GENOMIC DIGESTS
When Preparing Genomic DNA for Southern Blotting, How
Can You Determine if Complete Digestion Has Been
Obtained?
Southern blotting involves the digestion of genomic DNA, gel
electophoresis, blotting onto a membrane, and probing with a
labeled oligonucleotide. The restriction pattern after gel electrophoresis is usually a smear, which may contain some distinguishable bands when visualized by ethidium bromide staining.
It is often difficult to judge if the restriction digest has gone to completion or if degradation from star activity or nonspecific nuclease
contamination is occurring. A twofold serial digest of genomic
DNA enables a stable pattern, representing complete digestion, to
be distinguished from an incomplete or degraded pattern.
Complete digestion is indicated when a similar smear of DNA
appears in consecutive tubes of decreasing enzyme concentration
within the serial digest. If the tubes with high enzyme concentration show smears that contain fragments smaller than those seen
in tubes containing lesser enzyme, then it is likely that degradation is occurring. If the tube containing the most enzyme is the
only sample demonstrating a complete digest, then the subsequent
tubes (containing less enzyme) will demonstrate progressively
larger fragments. A uniformly banded pattern will not occur
in serial tubes unless the samples are all completely cut or
completely uncut (Figure 9.1).
If the size of the smear does not change even at the greatest
enzyme concentration, the digest may appear to have failed. A
second possibility is that the fragments are too large to be resolved
by standard agarose gel electrophoresis. Rare cutting enzymes
may produce fragments greater than 50 kb, may not cleave a
subset of sites due to methylation, or their recognition sequence
might be underrepresented in the genome being studied. Pulse
field gel electrophoresis must be used to resolve these fragments.
Tables listing the average size expected from digestion of different species’ DNA may be found in select suppliers’ catalogs.
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Robinson et al.
Figure 9.1 Testing for complete digestion of genomic
DNA. Twofold serial digest
using New England Biolabs
AvrII of Promega genomic
human DNA (cat. no. G304),
0.5 mg DNA in 50 ml NEB
Buffer 2 for 1 hour at 37°C.
AvrII added at 20 units and
diluted to 10 units, etc., with
reaction mix. The marker
NEB Low Range PFG
Marker (cat. no. N03050S).
Complete digestion is indicated by lanes 2–4. Photo
provided by Vesselin Miloushev and Suzanne Sweeney
New England Biolabs. Reprinted by permission of New
England Biolabs.
How Should You Prepare Genomic Digests for
Pulsed Field Electrophoresis?
Pulse field electrophoresis techniques including CHEF, TAFE,
and FIGE have made possible the resolution of DNA molecules
up to several million base pairs in length (Birren et al., 1989; Carle,
Frank, and Olson, 1986; Carle and Olson, 1984; Chu, Vollrath, and
Davis, 1986; Lai et al., 1989; Stewart, Furst, and Avdalovic, 1988).
The DNA used for pulsed field electrophoresis is trapped in
agarose plugs in order to avoid double-stranded breaks due to
shear forces. Protocol A has been used at New England Biolabs,
Inc. for the preparation and subsequent restriction endonuclease
digestion of E. coli and S. aureus DNA (Gardiner, Laas, and
Patterson, 1986; Smith et al., 1986). This protocol may be modified
as required for the cell type used.
Protocol A: Preparation of E. coli and S. aureus DNA
Cell Culture
1. Cells are grown under the appropriate conditions in 100 ml of
media to an OD590 equal to 0.8 to 1.0. The chromosomes are then
Restriction Endonucleases
245
aligned by adding 180 mg/ml chloramphenicol and incubating an
additional hour.
2. The cells are spun down at 8000 rpm at 4°C for 15 minutes.
3. The cell pellet is resuspended in 6 ml of buffer A at 4°C.
Alternatively 1.5 g of frozen cell paste may be slowly thawed in
20 ml of buffer A. Lysed cells from the thawing process are allowed
to settle and the intact cells suspended in the supernatant are
decanted and pelleted by centrifugation and washed once with
20 ml of buffer A. The pelleted cells are resuspended in 20 ml
of buffer A.
DNA Preparation and Extraction
1. The suspended cells are warmed to 42°C and mixed with an
equal volume of 1% low-melt agarose* in 1¥ TE at 42°C. For
S. aureus cells, lysostaphin is added to a final concentration of
1.5 mg/ml. The agarose solution may be poured into insert molds.
Alternatively, the agarose may be drawn up into the appropriate number of 1 ml disposable syringes that have the tips cut
off.
2. The molds or syringes are allowed to cool at 4°C for 10 minutes.
The agarose inserts are removed from the molds or extruded
from the 1 ml syringes.
3. A 12 ml volume of the agarose inserts is suspended in 25 ml of
buffer B (for E. coli), or 25 ml of buffer C (for S. aureus). Lysozyme
(for E. coli) or Lysostaphin (for S. aureus) is added to a final concentration of 2 mg/ml. The solution is incubated for two hours
at 37°C with gentle shaking. These solutions may also contain
20 mg/ml RNase I (DNase-free).
4. The agarose inserts are equilibrated with 25 ml buffer D for 15
minutes with gentle shaking. Replace with fresh buffer and repeat.
Replace with 25 ml of buffer D containing 2 mg/ml proteinase K.
This solution is incubated for 18 to 20 hours at 37°C with gentle
shaking.
5. The inserts are again subjected to 15 minutes gentle shaking with
25 ml of buffer E. Replace with fresh buffer and repeat. Then incubate for 1 hour in buffer E, with 1 mM Phenylmethylsulfonyl
fluoride (PMSF) to inactivate Proteinase K. As before, wash twice
more with buffer E.
6. The inserts are washed twice with 25 ml of buffer F. The inserts
are stored in buffer F at 4°C.
*Pulse field grade agarose should be used. The efficiency of the
restriction enzyme digestion may vary with different lots of other
low-temperature gelling agaroses.
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Robinson et al.
Digestion of Embedded DNA
Most restriction enzymes can be used to cleave DNA embedded in
agarose, but the amount of time and enzyme required for complete
digestion varies. Many enzymes have been tested for their ability to
cleave embedded DNA (Robinson et al., 1991).
1. Agarose slices containing DNA (20 ml) are equilibrated in 1.0 ml of
restriction enzyme buffer. The cylinders of agarose may be drawn
back up into the 1 ml syringes in order to accurately dispense
20 ml of the agarose. The solution is gently shaken at room
temperature for 15 minutes.
2. The 1 ml wash is decanted or aspirated from the agarose slice. The
insert slice is submerged in 50 ml of restriction enzyme buffer. The
appropriate number of units of the restriction enzyme with or
without BSA is added to the reaction mixture and digested for
a specific time and temperature as outlined by Robinson et al.
(1991).
3. Following the enzyme digestion, the inserts may be treated to
remove proteins using Proteinase K following the steps outlined
above. Alternatively, the slices may be loaded directly onto the
pulse field gel. Long-term storage of the endonuclease digested
inserts is accomplished by aspirating the endonuclease reaction
buffer out of the tube and submerging the insert in 100 ml of buffer
E at 4°C. Insert slices that have been incubated at 50°C during
the endonuclease digestion should be placed on ice for 5 minutes
before handling the sample for loading or aspirating the buffer.
List of Buffers
Buffer A Cell suspension buffer: 10 mM Tris-HCl pH 7.2 and
100 mM EDTA.
Buffer B Lysozyme buffer: 10 mM Tris-HCl pH 7.2, 1 M NaCl,
100 mM EDTA, 0.2% sodium deoxycholate, and 0.5% N-laurylsarcosine, sodium salt.
Buffer C Lysostaphin buffer: 50 mM Tris-HCl, 100 mM NaCl, and
100 mM EDTA.
Buffer D Proteinase K buffer: 100 mM EDTA pH 8.0, 1% N-laurylsarcosine, sodium salt, and 0.2% sodium deoxycholate.
Buffer E Wash buffer: 20 mM Tris-HCl pH 8.0 and 200 mM EDTA.
Buffer G Storage buffer: 1 mM Tris-HCl pH 8.0 and 5 mM EDTA.
What Are Your Options If You Must Create Additional Rare
or Unique Restriction Sites?
Cleavage at a single site in a genome may occur by chance
using restriction endonucleases or intron endonucleases, but the
Restriction Endonucleases
247
number of enzymes with recongition sequences rare enough to
generate megabase DNA fragments is relatively small. When
no natural recognition site occurs in the genome, an appropriate
sequence can be introduced genetically or in vitro via different
multiple step reactions.
Genetic Introduction
Recognition sites have been introduced into Salmonella
typhimurium and Saccharomyces cerevisiae genomes by site
specific recombination or transposition (Hanish and McClelland,
1991; Thierry and Dujon, 1992; Wong and McClelland, 1992).
Endogenous intron endonuclease recognition sites are found in
many organisms. In cases where restriction enzymes and intron
endonucleases cleave too frequently, it may be possible to use
lambda terminase. The 100 bp lambda terminase recognition site
does not occur naturally in eukaryotes. Single-site cleavage has
been demonstrated using lambda terminase recognition sites
introduced into the E. coli and S. cerevisiae genomes (Wang and
Wu, 1993).
Multiple-Step Reactions
The remainder of this discussion reviews multiple-step procedures that have been used to generate megabase DNA fragments.
Our intention is to provide a clear explanation of each procedure
and highlight some of the complexities involved. Providing
detailed protocols for each is beyond the scope of this chapter but
can be found in the references cited.
Increasing the complexity of multiple-step reactions decreases
the chances of success. Conditions needed for one step may not
be compatible with the next. All of the steps must function well
using agarose-embedded DNA as a substrate.
Altering Restriction Enzyme Specificity by DNA Methylation
DNA methylases can block restriction endonuclease cleavage
at overlapping recognition sites, decreasing the number of cleavable restriction sites and increasing the average fragment size
(Backman, 1980; Dobrista and Dobrista, 1980). Unique cleavage
specificities can be created by using different methylase/restriction
endonuclease combinations (Nelson, Christ, and Schildkraut,
1984; Nelson and Schildkraut, 1987). The following wellcharacterized, two-step reaction involves the restriction endonuclease NotI and a methylase (Gaido, Prostko, and Strobl, 1988;
Qiang et al., 1990; Shukla et al., 1991).
248
Robinson et al.
The NotI recognition site
5¢ . . . GCŸGGCCGC . . . 3¢
3¢ . . . CGCCGGŸCG . . . 5¢
will not cleave when methylation at the following cytosine occurs
in the NotI recognition site:
5¢ . . . GCGGCmCGC . . . 3¢
3¢ . . . CGCCGGCG . . . 5¢
or
5¢ . . . GCGGCCGC . . . 3¢
3¢ . . . CGmCCGGCG . . . 5¢
NotI sites that overlap the recognition site of the methylases M.
FnuDII, M. BepI, or M. BsuI can be modified as shown above.
These methylases recognize the following sequence:
5¢ . . . CGCG . . . 3¢
3¢ . . . GCGC . . . 5¢
They methylate the first cytosine in the 5¢ to 3¢ direction:
5¢ . . . mCGCG . . . 3¢
3¢ . . . GCGmC . . . 5¢
Now the subset of NotI sites that are preceded by a C or followed by a G will be resistant to subsequent cleavage by NotI.
Resistant sites
5¢ . . . CGCGGCCGC . . . 3¢
3¢ . . . GCGmCCGGCG . . . 5¢
or
5¢ . . . GCGGCmCGCG . . . 3¢
3¢ . . . CGCCGGCGC . . . 5¢
which are sites flanked by any of the following combinations, will
be cleaved by NotI:
5¢ . . . {A, G, T} GC Ÿ GGCCGC {A, C, T} . . . 3¢
3¢ . . . {T, C, A} CGCCGG Ÿ CG {T, G, A} . . . 5¢
This methylation reaction followed by NotI digestion statistically reduces the number of NotI sites by nearly half. The larger
Restriction Endonucleases
249
fragments produced may be more easily mapped using PFGE. A
table of other potentially useful cross-protections for megabase
mapping can be found in Nelson and McClelland (1992) and
Qiang et al. (1990). A potential problem is that certain methylation sites may react slowly allowing partial cleavage events (Qiang
et al., 1990).
DNA Adenine Methylase Generation of 8 to 12 Base-Pair
Recognition Sites Recognized by DpnI
DpnI is a unique restriction enzyme that recognizes and cleaves
DNA that is methylated on both strands at the adenine in its
recognition site (Lacks and Greenberg, 1975, 1977; Vovis, 1977).
DpnI recognizes the following site:
5¢ . . . G mA T C . . . 3¢
3¢ . . . C T mA G . . . 5¢
The adenine methylases M. TaqI (McClelland, Kessler, and
Bittner, 1984; McClelland, 1987), M. ClaI (McClelland, Kessler,
and Bittner, 1984; McClelland, 1987; Weil and McClelland, 1989),
M. MboII (McClelland, Nelson, and Cantor, 1985), and M. XbaI
(Patel et al., 1990) have been used to generate a DpnI recognition
site with the apparent cleavage frequency of a 8 to 12 base-pair
recognition sequence (Nelson and McClelland, 1992). The M.
TaqI/DpnI reaction is detailed below.
The M. TaqI recognition site
5¢ . . . TCGA . . . 3¢
3¢ . . . AGCT . . . 5¢
methylates the adenine on both strands of the above sequence to
produce
5¢ . . . T C G mA . . . 3¢
3¢.mA G C T . . . 5¢
Hemimethylated DpnI sites (in bold below) will be generated
when the sequence surrounding the site above is as follows:
5¢ . . . T C G mATC . . . 3¢
3¢ . . . mA G C TAG . . . 5¢
or
5¢ . . . G A T C GmA . . . 3¢
3¢ . . . C TmA G C T . . . 5¢
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Robinson et al.
The hemimethylated DpnI site is cleaved at a rate 60¥ slower
than the fully methylated site (Davis, Morgan, and Robinson,
1990). M. TaqI generates a fully methylated DpnI site when two
M. TaqI recognition sequences occur next to each other. The fully
methylated DpnI site is shown in bold below:
5¢. . . . TCG mA T C GmA . . . 3¢
3¢ . . . mAGC T mA G C T . . . 5¢
The apparent recognition site of the M. TaqI/DpnI reaction can
be simply represented by the eight base pairs 5¢ . . . TCGATCGA
. . . 3¢. The 10 base pair recognition site of the M. ClaI/DpnI reaction can be represented by the sequence 5¢ . . . ATCGATCGAT
. . . . 3¢. Notice that M. ClaI creates a DpnI site by a slightly different overlap than demonstrated by the M. TaqI reaction. The
M. ClaI/DpnI reaction has been demonstrated on a bacterial and
yeast genome (Waterbury et al., 1989; Weil and McClelland, 1989).
The M. XbaI/DpnI reaction can be represented by the 12 basepair sequence 5¢..TCTAGATCTAGA..3¢. This reaction has been
demonstrated on a bacterial genome (Hanish and McClelland,
1990).
We performed an extensive study of the M. TaqI/DpnI reaction.
The goal was to provide a mixture of the two enzymes that could
be used in a single-step reaction cleaving the eight base-pairs
5¢ . . . TCGATCGA . . . 3¢. Several potential problems concerning
M. TaqI were overcome. M. TaqI, a thermophile with a recommended assay temperature of 65°C, maintains greater than 50%
of its activity at 50°C. This is the maximum working temperature
for low-melt agarose. M. TaqI works well on DNA embedded in
agarose. Trace E. coli Dam methylase contamination was removed
from the recombinant M. TaqI by heat treatment at 65°C for
20 minutes. This is important because Dam methylase recognizes
5¢ . . . GATC . . . 3¢ and methylates the adenine creating DpnI
sites (Geier and Modrich, 1979). Two properties of the
DpnI make the reaction problematic. DpnI does not function
well on DNA embedded in agarose and hemimethylated sites are
cleaved slowly (Davis, Morgan, and Robinson, 1990; Nelson and
McClelland, 1992). A hemimethylated site generated at position
1129 on pBR322 could be completely cleaved with 60 units of
DpnI in one hour using the manufacturer’s recommended conditions. Partial digestion products were observed with greater than
5 units of DpnI.
As an alternative to agarose plugs, agarose microbeads (Koob
and Szybalski, 1992) should be prepared and the DNA embedded
Restriction Endonucleases
251
as described. The reduced diffusion distance offered by the agarose microbead matrix provides the enzyme with more effective
access to the embedded DNA substrate. DpnI should be diffused
into the microbeads by keeping the reaction mix on ice for at
least four hours prior to the 37°C incubation. To ensure complete
digestion, we suggest a range of DpnI concentrations from 1 to 10
units. Incubation time should not exceed two hours with DpnI
concentrations over 5 units.
Reducing the Number of Cleavable Sites via Blocking Agents
Coupled with a Methylase Reaction—Achilles’s Heel
Cleavage
Three classes of blocking reactions have been developed. All
three classes rely on the ability of a methylase to protect all but
one or more selected DNA sites from digestion by a restriction
endonuclease. We can summarize the methodology as follows:
A restriction endonuclease/methylase recognition site is
occupied by a blocking agent.
• The DNA is methylated, blocking subsequent cleavage at all
unoccupied sites.
• The blocking agent and methylase are removed.
• Restriction enzyme is added. Cleavage occurs only at previously blocked sites.
•
1. Achilles’ Heel Cleavage–DNA Binding Protein. A blocking reaction
using DNA binding proteins followed by restriction enzyme cleavage is termed “Achilles’ heel cleavage” (AC) (Koob, Grimes, and
Szybalski, 1988a). Unwanted cleavage can occur if the blocking
agent interacts with sites other than the one of interest, so blocking conditions should be optimized to minimize nonspecific interactions. These conditions must also allow the methylase to
function properly. If the blocking agent doesn’t stay bound to the
site for the duration of the methylation reaction, the blocking site
will be methylated, reducing the yield of the desired product.
Finally, all steps must work well on DNA substrates embedded
in agarose. The lac and lambda repressors were the first blocking reagents used in this type of reaction (Koob, Grimes, and
Szybalski, 1988b); phage 434 repressor (Grimes, Koob, and
Szybalski, 1990), and integration host factor (IHF) (Kur et al., 1992)
have also been used. Single-site cleavage has been attained using
the lac repressor site introduced into yeast and Escherichia coli
genomes (Koob and Szybalski, 1990).
Limitations to this strategy include the absence of natural
binding protein sites and the low frequency of restriction/methy252
Robinson et al.
lation sites. Binding protein sites have been engineered into
the target DNA, and degenerate sites containing the required
restriction/methylation sites have also been added (Grimes, Koob,
and Szybalski, 1990). However, modifications in the recognition
sequence of the binding protein can decrease the complex’s
half-life, allowing unwanted methylation at the AC site.
2. Achilles’ Heel Cleavage–Triple Helix Formation. The second
Achilles’ cleavage reaction uses oligonucleotide-directed triplehelix formation as a sequence specific DNA binding protein blocking agent (Hanvey, Schimizu, and Wells, 1990; Maher, Wold, and
Dervan, 1989). Pyrimidine oligonucleotides bind to homopurine
sites in duplex DNA to form a stable triple-helix structure. The
blocking reaction is followed by methylation, removal of the pyrimidine oligonucleotide and methylase, and cleavage by the restriction endonuclease. Single-site cleavage has been demonstrated on
yeast chromosomes by blocking with a 24 bp pyrimidine oligo,
(Strobel and Dervan, 1991a, 1992) and on human chromosome
4 using a 16 bp oligo (Strobel et al., 1991b). An advantage of this
method over the DNA binding protein AC is the increase in
frequency of sites. Insertion of the AC site into the genome is
not required. Relatively short purine tracts can be targeted using
sequence data. Degenerate probes can be used to screen for overlapping methylation/restriction endonuclease sites when suitable
sequence data are not available (Strobel et al., 1991b).
Reaction conditions for successful pyrimidine oligonucleotide
AC are complex (Strobel and Dervan, 1992). Triple helix formation using spermine can inhibit certain methylases, or precipitate
DNA in the low-salt reaction conditions required by some methylases. The narrow pH range for the protection reaction may not
be compatible with conditions required for efficient methylation.
Neutral or slightly acidic conditions promote highly stable triple
helices but reduce sensitivity to single base mismatches (Moser
and Dervan, 1987). Oligonucleotides that bind and protect mismatched sites allow nontarget restriction sites to remain unmethylated and subsequently cleaved. Increasing the pH from 7.2 to 7.8
can decrease the binding to similar sites (Strobel and Dervan,
1990). In higher pH reactions, the oligo does not stringently bind
to the intended target, allowing some methylation to occur at the
target site. The unwanted methylation reduces cleavage at the
Achilles’ site, lowering the yield of the desired DNA fragment.
3. Achilles’ Heel Cleavage–RecA-Assisted Restriction Endonuclease.
RecA-assisted restriction endonuclease (RARE) cleavage is the
most versatile of Achilles’ cleavage reaction discovered to date
Restriction Endonucleases
253
(Ferrin and Camerini-Otero, 1991; Koob and Szybalski, 1992). In
vitro studies indicate that in the presence of ATP, recA protein
promotes the strand exchange of single-stranded DNA fragments
with homologous duplex DNA. The three distinct steps in the
reaction are (1) recA protein binds to the single-strand DNA, (2)
the nucleoprotein filament binds the duplex DNA and searches
for a homologous region, and (3) the strands are exchanged (Cox
and Lehman, 1987; Radding, 1991). Stable triple-helix structures,
termed “synaptic complexes,” can be formed if the nonhydrolysable analog Adenosine 5¢-(g-Thio) triphosphate (ATPg S) is
substituted for ATP (Honigberg et al., 1985). The nucleoprotein
filament protects against methylation at a chosen site and is easily
removed exposing the AC site. Any duplex DNA stretch containing a restriction endonuclease/methylase recognition site, 15
nucleotides (nt) or longer in length, can be targeted (Ferrin and
Camerini-Otero, 1991). RARE cleavage has been used to generate single cuts in the E. coli genome by single-stranded oligonucleotides in the 30 nt range and on HeLa cell DNA with oligos in
60 nt range (Ferrin and Camerini-Otero, 1991). RecA-mediated
Achilles’ cleavage of yeast chromosomes using a 36-mer and 70mer has been demonstrated (Koob and Szybalski, 1992). YACs
(yeast artificial chromosomes) have been cleaved using nucleoprotein filaments in the 50 nt range (Gnirke et al., 1993).
Synaptic complex formation can also block cutting by a restriction endonuclease (Ferrin, 1995). Combined with the fact that
many restriction enzymes are active in the buffer used to form
these complexes, RARE can be applied to eliminate one of a pair
of identical restriction sites in a cloning vector. Partial digestion
has been applied to achieve a similar result, but this can fail if the
desired site is cut at a comparatively slow rate.
The complexities of the recA-mediated Achilles’ cleavage reaction include:
• A titration is required to find the exact ratio of recA to
oligonucleotide (Ferrin and Camerini-Otero, 1991; Koob and
Szybalski, 1992).
• Excess recA inhibits the methylation reaction.
• Complete hybridization of the oligonucleotide is required
for stable triplex formation.
• The nucleoprotein complex diffuses slowly into agarose;
microbeading is recommended when using this procedure.
• Nucleoprotein filaments produced with oligonucleotides
less than 40 nt may not be stable for the length of time required
254
Robinson et al.
for diffusion into agarose microbeads (Koob and Szybalski,
1992).
• RecA DNA-binding requires Mg2+.
• The methylases used must be free of contaminating
nucleases.
TROUBLESHOOTING
What Can Cause a Simple Restriction Digestion to Fail?
Faulty Enzyme or Problem Template Preparation?
If the suspect enzyme fails to digest a second or control target,
the titer of the enzyme activity should be measured by either
a twofold serial or a volumetric titration as described below
(procedures A and B).
If the titer assay indicates an active enzyme, and the enzyme
cleaves a control template but not the experimental DNA,
then an additional control digestion (procedure C) should be
performed to test for an inhibitor in the template preparation.
Often trans-acting inhibitors may be removed by the drop
dialysis protocol (procedure D) detailed below. Spin columns may
also be used to remove contaminants including primers, linkers,
and nucleotides (Bhagwat, 1992). A linearized plasmid containing
a single site may be used if cut and uncut samples are available as
markers.
As a matter of course, restriction enzyme activity should
be assayed by twofold serial titration if an enzyme has been
stored for a period longer than a year, an enzyme shipment was
delayed, or even if an enzyme was left on the bench overnight.
This simple assay may be used to test enzymes under nonoptimal conditions as well. Suppliers offer buffer charts that give
an indication of an enzyme’s expected activity in nonoptimal
buffers, and this information may be useful when the sample DNA
is in an alternative buffer due to a previous step or adapting
digests so that the DNA samples will be optimized for subsequent
steps.
Procedure A—Simple Twofold Serial Titer
Ideally the DNA should be the substrate on which the enzyme
was titered by the supplier. Lambda phage DNA or adenovirus
Type-2 DNA are common substrates used for enzyme titer. Any
DNA that contains several sites that produce a distinguishable
pattern may be applied.
Restriction Endonucleases
255
1.
2.
3.
4.
5.
6.
7.
8.
For the following experiment, make a total of 200 ml
of reaction mix. The reaction mix contains 1¥ reaction buffer,
1 mg DNA/50 ml reaction volume and BSA, if required. For this
example, the enzyme is supplied with a vial of 10¥ reaction
buffer and 10 mg/ml BSA. The final reaction mix requires 1¥
reaction buffer and 100 mg/ml BSA. Lambda DNA (commercially available at 500 mg/ml) is the substrate used to titer the
enzyme.
Add, in order:
a. 170 ml of distilled water
b. 20 ml of 10¥ buffer
c. 2 ml of 10 mg/ml BSA
d. 8 ml of 500 mg/ml Lambda DNA
Label six 1.5 ml microcentrifuge tubes (numbers 1–6). Pipette
50 ml of reaction mix into tube 1 and 25 ml of mix into the
remaining tubes.
Add 1 ml of restriction endonuclease to the first tube containing 50 ml of reaction mix. With the pipette set at 25 ml, mix by
gently pipetting several times.
From the 50ml reaction mix/enzyme, transfer 25 ml to the
second tube. This dilutes the enzyme concentration in half for
each subsequent tube.
Repeat step 4 until the final tube is reached. The final tube has
the most dilute enzyme, but indicates the highest titer. If the
final tube, in the following series, shows a complete digestion,
then the titer is at least 32,000 units/ml.
Cover each tube and incubate at the appropriate reaction
temperature for one hour.
The reaction is stopped by adding at least 10 ml stop
dye/50 ml reaction volume (50% 0.1 M EDTA, 50% glycerol,
0.05% bromophenol blue). The DNA fragments are resolved
by agarose gel electrophoresis, stained with ethidium bromide,
and visualized using ultraviolet light.
The titer is determined as follows:
Tube 1 complete: titer ≥1000 units/ml
Tube 2 complete: titer ≥2000 units/ml
Tube 3 complete: titer ≥4000 units/ml
Tube 4 complete: titer ≥8000 units/ml
Tube 5 complete: titer ≥16,000 units/ml
Tube 6 complete: titer ≥32,000 units/ml
The titer is based on the unit definition: 1 unit of restriction
enzyme digests 1 mg DNA to completion in 1 hour. If the digestion pattern from tube 1 is complete, then 1 ml of the enzyme
256
Robinson et al.
added contains at least 1 unit of activity. The concentration
1 unit/ml is the same as 1000 units/ml. With a dilution factor of 2,
a complete digestion pattern from tube 2 indicates that the
enzyme concentration is at least 2 ¥ 1000 units/ml = 2000 units/ml.
If tube 4 results in a complete digestion, and tube 5 results in a
partial banding pattern, the final titer of the enzyme may be conservatively estimated as 8000 units/ml. Similarly a more precise
serial dilution may be designed to evaluate the titer value between
8000 and 16,000 units/ml.
Procedure B—Volumetric Titration
The exact method will vary among enzyme manufacturers. You
should contact your supplier for the exact method if this information is not found in their catalog.
While not as convenient as serial titration for most benchtop
applications, most suppliers use volumetric titration to assay the
activity of the restriction endonucleases. This method may yield
more consistent results, especially when the enzyme stock is in
high concentration. Most volumetric titers require initial dilution
of the enzyme (often in 50% glycerol storage buffer) and the
use of substantial amounts of substrate DNA/reaction mix.
This method maintains constant enzyme addition to increasing
amounts of reaction mix volume, while keeping the concentration
of DNA substrate constant. The protocol may differ depending on
the concentration and dilution of the enzyme. This method is recommended when evaluating an enzyme sample to be ordered in
bulk amounts or for diagnostic applications where internal QC
evaluation is required.
Procedure C—Testing for Inhibitors
In a single vial with 1¥ reaction buffer, add 1 mg each of the
control and the experimental DNA. Add the restriction enzyme
and incubate at the recommended temperature and time. If there
is an inhibitor (often salt or EDTA), the mixed control substrate
will not cut.
Procedure D—Drop Dialysis (Silhavy, Berman,
and Enquist, 1984)
Many enzymes are adversely affected by a variety of contaminating materials in typical DNA preparations (minipreps, genomic
and CsCl2 preparations, etc.). The following drop dialysis method
has been successfully used to remove inhibitory substances (e.g.,
SDS, EDTA, or excess salt) from substrates intended for subsequent DNA manipulations. It is particularly effective for assuring
Restriction Endonucleases
257
complete cleavage of DNA by sensitive restriction endonucleases,
increasing the efficiency of ligation and preparation of templates
for DNA sequencing.
1a.
1b.
2.
3.
4.
258
For purification of genomic DNA, miniprep DNA, or DNA
used as a standard template for DNA sequencing: Phenol
extract, chloroform extract, and then alcohol precipitate the
DNA. Pellet the DNA in a microcentrifuge, pour off the
supernatant, and rinse the pellet with 70% ethanol. Dry
the pellet and resuspend it in 50 ml H20. (Proceed to step 2.)
For purification of templates for DNA sequencing of PCR
products: Phenol extract and then chloroform extract the
aqueous layer of the PCR reaction. Follow this with an
alcohol precipitation. Pellet the DNA by microcentrifugation, pour off the supernatant, and rinse the pellet with 70%
ethanol. Dry the pellet and resuspend it in 50 ml H2O. Alternatively, purify the PCR product through an appropriate spin
column, precipitate, and recover the DNA as described
above. PCR products that are not a single band on an agarose
gel should be gel-purified in low-melt agarose and then
treated with b-agarase I or a purification column technology.
When using b-agarase, treatment should be followed by
extraction, precipitation, and recovery, as described above.
When using a purification column, consult the manufacturer’s recommendations for the particular column
employed.
Pour 30 to 100 ml of dialysis buffer, usually double-distilled
water or 1¥ TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0), into
a petri plate or beaker.
Float a 25 mm diameter, Type VS Millipore membrane (cat.
no. VSWP 02500, MF type, VS filter, mean pore size =
0.025 mm, Millipore, Inc.) shiny side up on the dialysis buffer.
Allow the floating filter to wet completely (about 5 minutes)
before proceeding. Make sure there are no air bubbles
trapped under the filter.
Pipette a few microliters of the DNA droplet carefully onto
the center of the filter. If the sample has too much phenol or
chloroform, the drop will not remain in the center of the
membrane, and the dialysis should be discontinued until the
organics are further removed. In most cases this is performed
by alcohol precipitation of the sample. If the test sample
remains in the center of the membrane, pipette the remainder onto the membrane.
Robinson et al.
5.
6.
Cover the petri plate or beaker. Dialyze at room temperature. Be careful not to move the dish or beaker. Dialyze for
at least one hour and no more than four hours.
Carefully retrieve the DNA droplet with a micropipette.
Note that step 4 may be tricky for those with shaky hands or
poor hand-eye coordination. The filter has a tendency to move
briskly around the surface as you touch it with the pipette tip.
Practice with buffer droplets to master the technique before you
try using a valuable sample.
Dialysis against distilled water is also recommended, especially if
one is proceeding to another step where EDTA might be a problem.
The Volume of Enzyme in the Vial Appears Very Low.
Did Leakage Occur during Shipment?
Some enzymes (some offered at high concentration) may be
supplied in a very low volume and the vial may appear empty.
During shipment, the enzyme may be dispersed over most of the
interior surface of the vial or trapped just under the cap. Follow
the steps below to ensure that the enzyme volume is correct.
(Since the volume is very low, it is important to keep the entire
vial under ice or as cold as possible by working quickly.)
1.
Carefully check the exterior of the enzyme vial, noting any
signs of glycerol leakage.
2. Add the enzyme’s expected volume as water to an identical vial (for a counterbalance).
3. Briefly spin the enzyme vial in a microcentrifuge along with
the counterbalance.
4. With both vials on ice, estimate the volume of the enzyme
by comparison to that of the counterbalance.
The Enzyme Shipment Sat on the Shipping Dock for Two
Days. Is It Still Active?
Restriction enzymes are shipped on dry ice or gel ice packs,
depending on the supplier. When enzyme shipments arrive, there
should still be a good amount of dry ice left; or if shipped with ice
packs, these should still be cold, solid and not soft. For overnight
shipments, most suppliers include sufficient thermal mass to maintain proper shipping temperature for at least 36 hours. If the shipment was delayed en route, misplaced, or left in receiving for one
or more days, you should:
Restriction Endonucleases
259
Examine the contents, noting the integrity of the container.
If contents are still cold (but questionable in terms of actual
temperature), place a thermometer in the container, re-seal the
lid, and note the temperature after 10 minutes.
• After collecting details regarding the shipment’s ordering
information, contact the supplier. Customer service should
provide detailed information regarding the specific products in
question and, if warranted, shipping details for a replacement
order.
•
•
Generally, if the enzyme package is still cold to the touch, most
enzymes should be completely active, even if the 10¥ buffers have
recently thawed. Due to their salt content, the concentrated
buffers would be liquid even at 0°C. If the enzyme is required for
use immediately and no alternative source is available, the enzyme
may be tested for activity by serial titration, as described above.
Also bear in mind that many enzymes retain their activity after a
16 hour incubation at room temperature (McMahon, M., and
Krotee, S., unpublished observation).
Analyzing Transformation Failures and Other Multiple-Step
Procedures Involving Restriction Enzymes
A restriction digest is rarely the ultimate step of a research
procedure, but instead an early (and essential) reaction within a
multiple-step process, as in the case of a cloning experiment.
Therefore, when troubleshooting restriction enzymes, and more so
than other reagents, it is essential to objectively list all the feasible explanations for failure as noted in step 2 of the troubleshooting strategy discussed in Chapter 2, “Getting What You
Need From A Supplier.” The following discussion illustrates the
importance of identifying and investigating all the possible causes
of what appears to be a restriction enzyme failure.
If background levels are high after transformation, the enzyme
activity should be checked. Alternatively, the vector may have
ligated to itself. If the vector had symmetric ends, were the 5¢ phosphates removed by dephosphorylation? Was the effectiveness of
the dephosphorylation proved? Incomplete vector digestion
might be caused by contaminants in the DNA preparation, incompatible buffer, insufficient restriction enzyme, or sites that are
located adjacent to each other. If the vector had two different
termini, was the success of both digestions verified by recircularization experiments?
Exonuclease contamination in the restriction enzyme or
DNA preparation can prevent insert ligation, but ligation might
260
Robinson et al.
proceed if the ends are blunted by the exonuclease. In this scenario the restriction site would be lost and the reading frame
shifted. Phenol chloroform extraction followed by ethanol precipitation will remove exonuclease from DNA preparations.
Check the restriction enzyme quality control data for exonuclease, ligation, and blue-white selection. Do not extend the digestion time if an exonuclease problem is suspected.
DNA preparations can contain contaminants that inhibit
ligation as well as restriction endonuclease digestion, and the use
of very dilute DNA solutions can amplify inhibition. Higher stock
vector and insert concentrations are preferable because less of
the final reaction volume comes from the DNA solution. If the
DNA is stored in Tris-EDTA, the EDTA may inhibit the ligation
or restriction digest. Using dilute DNA solutions gives less flexibility when choosing the molar ratio of insert to vector and final
DNA concentration of the reaction; both parameters directly
affect the quantity of desirable products produced in the ligation
reaction.
Failed ligation can occur if the molar ratio of insert to vector is
not sufficient. A molar ratio of 3 : 1 insert to vector should be used
for asymmetric ligations and symmetric ligations with small
inserts. Symmetric ligations with inserts greater than 800 bp should
use 8 mg/ml insert to 1 mg/ml vector (Revie, Smith, and Yee, 1988).
In general, the vector concentration should be kept at 1 mg/ml.
Total DNA concentration should be kept to 6 mg/ml or less
(Bercovich, Grinstein, and Zorzopulos, 1992). Blunt ends are
treated as symmetric, and overnight ligation at 16°C is recommended. The addition of 7% PEG 8000 can also stimulate ligation. Single-base overhangs are more difficult to ligate than blunt
ends; overnight ligation at 16°C using concentrated ligase is also
suggested here. Even so, less than 20% ligation is seen for Tth111I
under these conditions. Filling in the 5¢ single-base overhang with
Klenow resulting in a blunt end will increase ligation to about
40% (Robinson, D., unpublished observation).
Transformants containing only deletions indicate problems
with ligation or dephosphorylation. Blunt end ligation of a PCR
product made with unphosphorylated primers into a dephosphorylated vector will result in a failed ligation, although competent
cells will take up some linear molecules. Cells can scavenge the
antibiotic resistance gene used for selection, and the scavenged
gene is normally found on a vector containing a deletion. The
miniprep DNA from the transformants will often run smaller than
the control linearized vector.
Faulty DNA ligase, a reaction buffer lacking ATP, and the addiRestriction Endonucleases
261
tion of too much ligation mix to the competent cells can result in
low colony count. An antibiotic in the plate that doesn’t match the
resistance gene within the vector or leaky expression of a toxic
protein can kill competent cells, which could mimic a restriction
enzyme failure. Cells can be tested by transformation using uncut
vector. In addition, as restriction enzymes are excellent DNA
binding proteins, they can remain bound to DNA termini and
inhibit ligation. Active restriction enzyme can recleave ligated
DNA. Often, after incubation, this effect may be minimized by
either heating the reaction to 65°C or proceeding with an alternative purification step.
Failure at any one of the many steps of a cloning experiment
can give the impression of a restriction enzyme failure. The same
principle holds true for the many other applications that involve
restriction enzymes.
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